The broad objectives of this project are (1) to localize the molecular determinants responsible for activation of voltage-gated Na+ channels, and (2) to explore the possibility that the activation kinetics of cardiac Na+ channels contribute to local anesthetic-induced cardiotoxicity. The voltage-dependence of activation at cardiac and skeletal muscle Na+ channels is clearly different, yet no study has defined the molecular properties responsible for this difference. The initial focus of this application is to identify specific amino acid differences at cardiac and skeletal muscle Na+ channels that may impart their unique activation kinetics. The more depolarized activation range of skeletal muscle Na+ channels suggests that the outward movement of the voltage sensing S4 segments is more impeded compared to the outward movement of S4 segments in cardiac Na+ channels. Because the amino acid sequences of S4 voltage sensors in human cardiac (hH1) and rat skeletal muscle (mul) Na+ channels are virtually identical, the hypothesis to be tested is whether molecular interactions between residues in the voltage sensing S4 segments and residues in other segments can explain the differences in activation. The amino acid sequences of hH1 and mul Na+ channels differ at at least eight potentially important sites in other segments of the four domain regions. Cation-pi electron interactions or charge-charge interactions between the charged residues in the S4 segments and residues in the other segments could impede outward movement of mul S4 segments and thus explain the more depolarized activation range. Among the experiments designed to investigate this possibility, a mul residue will be substituted for the native residue at the same site in hHl channels in an attempt to alter the activation kinetics of the hHl isoform. Steady-state block of wild-type and mutant hHl channels by bupivacaine, a cardiotoxic local anesthetic, will be investigated under whole-cell voltage clamp conditions. The roles of these specific mutations in determining steady-state block will be further investigated using simulations designed to predict block. Relationships between the kinetic parameters and the state-dependent affinities of the channels will be used to revise models of local anesthetic block at cardiac Na+ channels. A model that incorporates possible roles for both channel activation and channel inactivation may be necessary to more fully explain the enhanced sensitivity of cardiac tissue to circulating local anesthetics.